WO2007107714A2 - Corrélats cliniques - Google Patents

Corrélats cliniques Download PDF

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Publication number
WO2007107714A2
WO2007107714A2 PCT/GB2007/000934 GB2007000934W WO2007107714A2 WO 2007107714 A2 WO2007107714 A2 WO 2007107714A2 GB 2007000934 W GB2007000934 W GB 2007000934W WO 2007107714 A2 WO2007107714 A2 WO 2007107714A2
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WIPO (PCT)
Prior art keywords
cells
infection
ifn
individual
pathogen
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PCT/GB2007/000934
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English (en)
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WO2007107714A3 (fr
WO2007107714A8 (fr
Inventor
Ajit Lalvani
Kerry Millington
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Ajit Lalvani
Kerry Millington
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Application filed by Ajit Lalvani, Kerry Millington filed Critical Ajit Lalvani
Priority to US12/293,249 priority Critical patent/US8765366B2/en
Priority to AU2007228598A priority patent/AU2007228598A1/en
Priority to PL07712923T priority patent/PL2005182T3/pl
Priority to ES07712923T priority patent/ES2423479T3/es
Priority to DK07712923.7T priority patent/DK2005182T3/da
Priority to EP07712923.7A priority patent/EP2005182B1/fr
Priority to JP2008558905A priority patent/JP5258584B2/ja
Publication of WO2007107714A2 publication Critical patent/WO2007107714A2/fr
Publication of WO2007107714A3 publication Critical patent/WO2007107714A3/fr
Publication of WO2007107714A8 publication Critical patent/WO2007107714A8/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/55—IL-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates methods of monitoring mycobacterial infections, such as Mycobacterium tuberculosis infection and kits for use in such methods.
  • markers of infection status of an individual Use of such markers allows treatment, prevention and control of the relevant disease.
  • monitoring the status of an individual infected with tuberculosis (TB) is essential for the treatment, prevention and control of this resurgent disease.
  • Mycobacterial load cannot be measured directly and there is currently no biomarker reflecting mycobacterial load.
  • Mycobacterium tuberculosis is often difficult to culture from patients with active TB, and impossible to culture from healthy latently infected people. Therefore an immune-based diagnostic test indicating the status of the Mycobacterium tuberculosis infection would be very useful in a number of clinical scenarios guiding the treatment, prevention and control of this infection. For example, differentiating between active and latent infection, monitoring efficacy of anti-tuberculosis therapy of active and latent TB, monitoring efficacy of therapeutic vaccines and new drugs and detecting early progression to active disease.
  • ESAT-6 early secretory antigen target-6
  • CPFlO culture filtrate protein 10
  • ESAT-6 and CPFlO are strong targets of the cellular immune response in animal models, tuberculosis patients and contacts and so may be used in specific T cell-based blood tests.
  • the present inventors have shown that the profile of T-cell-derived IFN- ⁇ and IL-2 secretion correlates with different clinical outcomes of intracellular pathogen infection.
  • the invention thus provides an indirect immunological biomarker of intracellular pathogen load and status in vivo which can be used to predict, determine and monitor clinical outcome as well as monitor the effect of therapeutic or vaccine intervention on infection.
  • the present invention provides: a method for assessing an intracellular pathogen infection, said method comprising:
  • Figure 1 shows the IFN- ⁇ and IL-2 cytokine profile of CD4+ T cells in active tuberculosis and during and after anti-tuberculosis therapy. The percentages shown indicate the relative proportions of CD4+ cells producing IFN- ⁇ and IL-2 in the enriched fraction with background levels of non-specific cytokine production subtracted.
  • ESAT-6 and CFP-10-specific EFN- ⁇ and IFN- ⁇ /IL-2-secreting CD4+ T cells co-dominate in active tuberculosis, in whom bacterial load is high (A, B), whilst EFN- ⁇ /IL-2-secreting T cells predominate during and after treatment which eradicated viable bacilli in vivo, with the loss of IFN- ⁇ only secreting CD4+ T cells and the appearance of IL-2-secreting CD4+ T cells (C, D).
  • This data was reproducibly observed in 4 active tuberculosis cases and in 9 follow-up samples from 5 patients 3 months into treatment and thereafter.
  • Figure 2 shows the proportion and frequency of ESAT-6 and CFP-10-specific
  • CD4 + T cell IFN- ⁇ (white bars/symbols), IFN- ⁇ /IL-2 (grey bars/symbols) and EL-2 (black bars/symbols) responses in active TB compared to follow up time points (during and after treatment).
  • the proportion of ESAT-6 and CFP-10-specific CD4 + T cells secreting IFN- ⁇ and/or IL-2 in the enriched fraction (A) and frequencies of these cells as . determined after magnetic enrichment of IFN- ⁇ and EL-2-secreting cells (B) are shown.
  • the horizontal lines indicate the median positive response.
  • the P values indicate whether there is a statistical difference between active TB and follow up.
  • Figure 3 shows the IFN- ⁇ and IL-2 cytokine profile of CD4+ T cells in self- healed tuberculosis.
  • PBMC were stimulated with ESAT-6 and CFP-10 and enriched for IFN- ⁇ and IL-2-secreting cells as for Figure 1.
  • EFN- ⁇ , IFN- ⁇ /IL-2 and IL-2-secreting CD4+ T cells were observed in self-healed participants in whom viable bacilli persist.
  • Figure 4 summarises the observed cytokine profiles in different outcomes of tuberculosis infection.
  • FIG. 5 shows potential applications of this invention.
  • SEQ ED NO: 1 is the amino acid sequence of the Mycobacterium tuberculosis antigen ESAT-6.
  • SEQ ED NO: 2 is the amino acid sequence of the Mycobacterium tuberculosis antigen CPFlO. Detailed Description of the Invention
  • the invention provides a method of assessing an intracellular pathogen infection and/or monitoring an intracellular pathogen infection in an individual, comprising determining whether the individual has (a) T-cells that secrete IFN- ⁇ only, (b) T-cells that secrete IL-2 only or (c) T-cells that secrete both EFN- ⁇ and IL-2 in response to an intracellular pathogen antigen and optionally determining any change in this cytokine profile.
  • the intracellular pathogen may be a virus, bacterium or parasite.
  • the virus is typically CMV, hepatitis A, hepatitis C, EBV or HSV.
  • the pathogen may be Leishmaniasis, leprosy or Listeria.
  • the infection is a mycobacterial infection, such as Mycobacterium tuberculosis.
  • the method may be used to determine the pathogen load, predict development and/or reactivation of disease, monitor anti-pathogen therapy, evaluate the efficacy of anti-pathogen drugs or vaccines or to determine whether anti-pathogen therapy results in a sterilising cure.
  • the method may be used to determine and/or monitor whether a patient has an active infection, latent infection, infection or disease which has spontaneously cleared or is clearing, infection or disease which has cleared or is clearing without medical intervention, or infection or disease which has been or is being cured.
  • a method of the invention typically comprises:
  • the profile of (a), (b) and (c) indicates the status of the infection.
  • the invention provides a method for determining pathogen load in an individual, wherein the presence and/or proportion of (a) is positively correlated with pathogen load and the presence and/or proportion of (b) is negatively correlated with pathogen load.
  • the individual is typically a mammal, preferably a human.
  • the human may have an active or latent mycobacterial infection, or may have had such an infection recently.
  • the human may test positive or negative in a Mantoux test.
  • the human may be at risk of infection (such as mycobacterial infection), typically for socio-economic reasons or may have a genetic or acquired predisposition to the infection.
  • the mycobacterial infection may be caused by any mycobacteria.
  • the mycobacterial infection is caused by Mycobacterium tuberculosis.
  • the human may be a known or suspected contact who has been exposed to or may have been exposed to Mycobacterium tuberculosis.
  • the exposure is to pulmonary tuberculosis, such as 'open' pulmonary tuberculosis which is sputum A.F.B. (acid-fast bacillus) smear positive.
  • the contact may be someone whose exposure is a household, work place (such as a health care worker) or prison exposure (such as a prisoner).
  • the exposure may have resulted from residing in a country with high prevalence of TB, and diagnostic testing after emigration to a country with a low prevalence of TB.
  • the contact may be an immigrant.
  • the human for example who has a known or suspected recent or remote exposure
  • may be healthy might have a co-infection or chronic condition or be on therapeutic agents putting them at a higher risk of developing active TB and/or which may make TB infection harder to diagnose.
  • examples include HIV infected individuals, individuals taking immunosuppressants (e.g. corticosteroids, azathioprine and anti-TNF- ⁇ agents, such as infliximab, and cancer therapy), hemodialysis patients, organ transplant recipients, diabetics and very young children (aged under 5 years old, particularly under 2 years old).
  • immunosuppressants e.g. corticosteroids, azathioprine and anti-TNF- ⁇ agents, such as infliximab, and cancer therapy
  • hemodialysis patients hemodialysis patients
  • organ transplant recipients diabetics and very young children (aged under 5 years old, particularly under 2 years old).
  • the T-cells which recognise the antigen in the method are generally T-cells which have been pre-sensitised in vivo to antigen from the pathogen. These antigen- experienced T-cells are generally present in the peripheral blood of a host which has been exposed to the pathogen at a frequency of 1 in 10 6 to 1 in 10 3 peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the T-cells may be CD4 and/or CD8 T-cells.
  • the T-cells can be contacted with the antigen in vitro or in vivo, preferably in vitro in a sample from the individual.
  • the T-cells which are contacted in the method are taken from the host in a blood sample, although other types of samples which contain T-cells can be used.
  • the sample may be added directly to the assay or may be processed first.
  • the processing may comprise diluting of the sample, for example with water, buffer or media.
  • the sample is diluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10 fold.
  • the processing may comprise separation of components of the sample.
  • mononuclear cells MCs
  • the MCs will comprise the T-cells and antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • the APCs present in the separated MCs can present the peptide to the T-cells.
  • only T-cells such as only CD4 T-cells, can be purified from the sample.
  • PBMCs, MCs and T-cells can be separated from the sample using techniques known in the art, such as those described in Lalvani et al (1997) J.Exp. Med. 186, p859-865.
  • the T-cells used in the assay are in the form of unprocessed or diluted samples, are freshly isolated T-cells (such as in the form of freshly isolated MCs or PBMCs) which are used directly ex vivo, i.e. they are not cultured before being used in the method or are thawed cells.
  • the T-cells can be cultured before use, for example in the presence of the antigen, and generally also exogenous growth promoting cytokines.
  • the antigen is typically present on the surface of APCs, such as the APC used in the method. Pre-culturing of the T-cells may lead to an increase in the sensitivity of the method.
  • the T-cells can be converted into cell lines, such as short term cell lines (for example as described in Ota et al (1990) Nature 346, pl83-187).
  • the APC which is typically present in the method may come from the same host as the T-cell or from a different host.
  • the APC may be a naturally occurring APC or an artificial APC.
  • the APC is a cell which is capable of presenting the antigen to a T-cell. It is typically a B-cell, dendritic cell or macrophage. It is typically separated from the same sample as the T-cell and is typically co-purified with the T-cell. Thus the APC may be present in MCs or PBMCs.
  • the APC is typically a freshly isolated ex vivo cell or a cultured cell. It may be in the form of a cell line, such as a short term or immortalised cell line.
  • the APC may express empty MHC class II molecules on its surface.
  • the antigen is added to an assay comprising T-cells and APCs.
  • the antigen may be coated on an inner aspect of the container e.g. a tube into which the blood is drawn from the patient; alternatively, the antigen may be added to a container that already contains the T-cells and APCs.
  • the T-cells and APCs in such an assay could be in the form of MCs.
  • APCs are not required.
  • Analogues which mimic the original antigen bound to a MHC molecule are an example of such an antigen.
  • the antigen is provided to the APC in the absence of the T-cell.
  • the APC is then provided to the T-cell, typically after being allowed to present the antigen on its surface.
  • the antigen may have been taken up inside the APC and presented, or simply be taken up onto the surface without entering inside the APC.
  • 10 5 to 10 7 preferably 2.5xlO 5 to 10 6 PBMCs are added to each assay.
  • peptide is added directly to the assay its concentration is from 10 "1 to 10 3 ⁇ g/rnl, preferably 0.5 to 50 ⁇ g/ml or 1 to lO ⁇ g/ml.
  • the length of time for which the T cells are incubated with the antigen is from 4 to 24 hours (preferably 5 to 18 hours) for effector T-cells or for more than 24 hours for central memory cells.
  • the length of time for which the T cells are incubated with the antigen is from 4 to 24 hours (preferably 5 to 18 hours) for effector T-cells or for more than 24 hours for central memory cells.
  • any suitable antigen may be used in a method of the invention.
  • the antigen is a mycobacterial or M. tuberculosis antigen.
  • the antigen may be in the form of a naturally occurring protein which is recognised by a T-cell.
  • Suitable M. tuberculosis antigens include ESAT-6 and CFP-10. The amino acid sequences of these antigens are shown in SEQ ID NOs: 1 and 2, respectively.
  • the antigen may be a fragment (such as a peptide) and/or homologue of a naturally occurring protein which is recognised by a T-cell that recognises the natural T- cell epitope sequence.
  • the antigen may also be an analogue which mimics the epitope of the naturally occurring protein bound to a MHC molecule.
  • IL-2 or IFN- ⁇ can typically be detected by allowing them to bind to a specific capture agent which may be immobilised on a support such as a plate, bead or the cytokine-secreting cell itself, and then measuring the presence of the specific binding agent/IL-2 complex and/or the specific binding agent/IFN- ⁇ complex typically with a second binding detection agent.
  • a washing step can be incorporated to remove material which is not specifically bound to the capture agent.
  • the second agent binds IFN- ⁇ or IL-2 at a site which is different from the site which binds the first agent.
  • the second agent may be directly conjugated to an enzyme such as alkaline phophatase, or fluorescent label or may comprise a biotin moiety to be detected by a third agent comprising strepavidin, which is directly conjugated to an enzyme or fluorescent label.
  • the conjugated enzyme then changes colour of a reagent.
  • the agent is preferably an antibody, mono- or polyclonal. Antibodies to IL-2 and IFN- ⁇ are commercially available, or can be made using standard techniques.
  • the preferred method employed to detect EFN- ⁇ and/or IL-2 will be based on sandwich immunoassays detecting the frequency of cytokine-secreting cells such as colour or fluorescent ELISpot, limited dilution assays, intracellular cytokine staining and cytokine secretion assays with or without enrichment of cytokine-secreting cells as pioneered by Miltenyi Biotec.
  • the amount of cytokine secreted can be measured for example by an ELISA based system such as the whole blood Quantiferon® system with the capture antibody immobilised on a plate, and its modifications (for example as available from Cellestis) or Luminex® suspension array technology using Beadlyte® kits with the capture antibody immobilised on a bead. Cytokine mRNA expression can also be measured with assays such as RT-PCR.
  • the detection system which is used is the ex-vivo ELISpot assay described in WO 98/23960.
  • IFN- ⁇ secreted from the T-cell is bound by a first IFN- ⁇ specific antibody which is immobilised on a solid support.
  • the bound IFN- ⁇ is then detected using a second IFN- ⁇ specific antibody which is labelled with a detectable label.
  • a labelled antibody can be obtained from MABTECH (Stockholm, Sweden).
  • Other detectable labels which can be used are discussed below.
  • the methods of the invention allow the determination of whether pathogen antigen-responsive T-cells that produce IFN- ⁇ , IL-2 or both IFN- ⁇ and IL-2 are each present in an individual.
  • each type of antigen-responsive T-cell may also be determined.
  • the presence or absence of each type of T-cell enables the state of the infection in the individual and/or the mycobacterial load to be determined.
  • the presence of IFN- ⁇ only secreting cells and cells which secrete both IFN- ⁇ and IL-2 occurs during states of high antigen load.
  • antigen-specific T-cells stop secreting IFN- ⁇ only, predominantly secrete both IFN- ⁇ and IL-2 and newly secrete IL-2 only.
  • IFN- ⁇ only secreting cells are positively correlated with antigen load and IFN- ⁇ /IL-2 secreting cells are negatively correlated with antigen load.
  • the profile of the three types of antigen-responsive T-cells may be used to determine pathogen load in the individual. Where the individual has a latent mycobacterial infection, but is not showing disease symptoms assessing and/or monitoring the profile of IFN- ⁇ , IL-2 and IL-2/IFN- ⁇ secreting cells will be predictive of the risk of reactivation. The presence of cells secreting IFN- ⁇ only or IFN- ⁇ /IL-2 typically indicates that the individual has a latent infection associated with a risk of progression to active tuberculosis.
  • the number or proportion of cells secreting EFN- ⁇ only may positively correlate with the risk of progression or reactivation, which is also strongly influenced by several host factors including for example, but not limited to, age, concomitant illness, HIV co-infection, iatrogenic immunosuppression and how recent the infection is.
  • the number or proportion of cells secreting IL-2 only with or without the presence of cells secreting EFN- ⁇ and IL-2, may inversely correlate with the risk of progression or reactivation and a relatively high number of IL-2-secreting cells may indicate a very low risk of reactivation.
  • the method may be used to monitor the progress of anti-pathogen (preferably mycobacterium) therapy in active or latent infection in an individual.
  • a decrease in the number of (or relative amount of) IFN- ⁇ only secreting cells and/or an increase in the number of (or relative amount of) IL-2 secreting cells indicates that the therapy is effective.
  • an increase in the predominance of cells that secrete both IFN- ⁇ and IL-2 is an indication that the therapy is working.
  • the method may be used to evaluate the efficacy of known anti-pathogen drugs or vaccines or to test the efficacy of potential new anti-pathogen drugs or vaccines.
  • a decrease in the number of (or relative amount of) IFN- ⁇ only secreting cells and/or an increase in the number of (or relative amount of) IL-2 secreting cells indicates that the therapy is efficacious.
  • an increase in the predominance of cells that secrete both IFN- ⁇ and IL-2 is also a positive indication of efficacy.
  • the profile of the three antigen-responsive cell types may be examined following infection to determine the effectiveness of the vaccine.
  • a profile associated with low pathogen load indicates that the vaccine is effective and a profile associated with high pathogen load is less promising.
  • the individual tested in a method of the invention may have received an anti-pathogen drug or vaccine.
  • the method may further comprise comparing the status of said infection to the previously determined status of said infection in said individual, thereby monitoring the effectiveness of said anti-pathogen drug or vaccine in said individual.
  • the individual may have received a test anti-pathogen drug or vaccine.
  • the method may further comprise comparing the status of said infection to the previously determined status of said infection in said individual, thereby determining the efficacy of said test drug or vaccine.
  • the invention also provides a kit for carrying out the methods of the invention comprising means for detecting cells that secrete IFN- ⁇ , IL-2 or both EFN- ⁇ and IL-2 in response to a pathogen (preferably mycobacterial) antigen.
  • the kit may optionally include instructions for correlating the profile of IFN- ⁇ , IL-2 and/or IFN- ⁇ and IL-2 secreting cells to the status of the infection.
  • the instructions may be for correlating the detection of IFN- ⁇ , IL-2 and/or IFN- ⁇ and IL-2 secreting cells to the pathogen load.
  • the means to detect recognition allows or aids detection based on the techniques discussed above.
  • the means may allow detection of IFN- ⁇ and IL-2 secreted by the T-cells after recognition.
  • the kit may thus additionally include a specific binding agent for each of IFN- ⁇ and IL-2, such as an antibody.
  • the agent is typically immobilised on a solid support. This means that after binding the agent the
  • IFN- ⁇ and/or IL-2 will remain in the vicinity of the T-cell that secreted it.
  • spots' of IFN- ⁇ /agent and/or IL-2 /agent complex are formed on the support, each spot representing a T-cell which is secreting IFN- ⁇ and/or IL-2. Quantifying the spots, and typically comparing against a control, allows determination of the relative numbers of cells that secrete IFN- ⁇ , IL-2 or both IFN- ⁇ and IL-2.
  • the kit may also comprise a means to detect the IFN- ⁇ /agent arid/or IL-2 /agent complexes.
  • a detectable change may occur in the agent itself after binding IFN- ⁇ and/or IL-2, such as a colour change.
  • a second agent directly or indirectly labelled for detection may be allowed to bind the IFN- ⁇ /agent and/or IL-2 /agent complex to allow the determination of the spots.
  • the second agent may be specific for IFN- ⁇ or IL-2, but binds a different site on IFN- ⁇ or IL-2 than the first agent.
  • the immobilised support may be a plate with wells, such as a microtitre plate.
  • Each assay can therefore be carried out in a separate well in the plate.
  • the kit may additionally comprise medium for the T-cells, detection agents or washing buffers to be used in the detection steps.
  • the kit may additionally comprise reagents suitable for the separation from the sample, such as the separation of PBMCs or T-cells from the sample.
  • the kit may be designed to allow detection of the T-cells directly in the sample without requiring any separation of the components of the sample.
  • the kit may also comprise controls, such as positive or negative controls.
  • the positive control may allow the detection system to be tested. Thus the positive control typically mimics recognition of the peptide in any of the above methods.
  • the positive control is IFN- ⁇ and/or
  • the kit may also comprise a means to take a sample containing T-cells from the human, such as a blood sample.
  • the kit may comprise a means to separate mononuclear cells or T cells from a sample from the individual.
  • Example 1 IFN- ⁇ and IL-2 cytokine profile of CD4+ T cells in active tuberculosis and during and after anti-tuberculosis therapy Frozen PBMC were stimulated with M. tuberculosis-specific ESAT-6 or CFP-10 peptide pools (15-mers overlapping by 10 amino-acids) for 5 h at 37°C. Secreted IFN- ⁇ and IL-2 was then caught on the surface of the secreting cell for 45 min at 37 0 C followed by enrichment for IFN- ⁇ and IL-2-positive cells with the IFN- ⁇ (APC) and IL- 2 (PE) cell enrichment and detection kits according to the manufacturer (Miltenyi Biotec Ltd).
  • APC IFN- ⁇
  • PE IL- 2
  • PBMCs were stained with anti-CD4, anti-IFN- ⁇ , anti-IL-2 antibodies, and PerCP-conjugated anti-CD14, PerCP-conjugated anti-CD19 antibodies to exclude monocytes and B cells respectively, and Via-Probe to exclude dead cells. Plots were gated on CD4+ CD14- CD19- Via-Probe- cells.
  • Example 2 IFN- ⁇ and IL-2 cytokine profile of CD4+ T cells in self-healed tuberculosis PBMC were stimulated with ESAT-6 and CFP-IO and enriched for IFN- ⁇ and IL-2-secreting cells as described in Example 1. EFN- ⁇ , IFN- ⁇ /IL-2 and IL-2-secreting CD4+ T cells were observed in self-healed participants in whom viable bacilli persist. The self -healed participants are an at-risk group of reactivation of disease.

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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode d'évaluation et/ou de surveillance d'une infection pathogène intracellulaire chez un individu, cette méthode consistant à déterminer si l'individu a (a) des cellules T qui sécrètent seulement de l'IFN-γ, (b)des cellules T qui sécrètent seulement de l'IL-2 ou (c) des cellules T qui sécrètent à la fois de l'IFN-γ et de l'IL-2 en réponse à un antigène pathogène intracellulaire, la méthode consistant optionnellement à déterminer tout changement dans ce profil cytokine.
PCT/GB2007/000934 2006-03-17 2007-03-16 Corrélats cliniques WO2007107714A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US12/293,249 US8765366B2 (en) 2006-03-17 2007-03-16 Clinical correlates
AU2007228598A AU2007228598A1 (en) 2006-03-17 2007-03-16 Clinical correlates
PL07712923T PL2005182T3 (pl) 2006-03-17 2007-03-16 Kliniczne korelaty
ES07712923T ES2423479T3 (es) 2006-03-17 2007-03-16 Correlatos clínicos
DK07712923.7T DK2005182T3 (da) 2006-03-17 2007-03-16 Kliniske korrelater
EP07712923.7A EP2005182B1 (fr) 2006-03-17 2007-03-16 Correlats cliniques
JP2008558905A JP5258584B2 (ja) 2006-03-17 2007-03-16 臨床的相関

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GBGB0605474.6A GB0605474D0 (en) 2006-03-17 2006-03-17 Clinical correlates
GB0605474.6 2006-03-17

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WO2007107714A2 true WO2007107714A2 (fr) 2007-09-27
WO2007107714A3 WO2007107714A3 (fr) 2007-11-15
WO2007107714A8 WO2007107714A8 (fr) 2009-07-16

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US (1) US8765366B2 (fr)
EP (1) EP2005182B1 (fr)
JP (1) JP5258584B2 (fr)
AU (1) AU2007228598A1 (fr)
DK (1) DK2005182T3 (fr)
ES (1) ES2423479T3 (fr)
GB (1) GB0605474D0 (fr)
PL (1) PL2005182T3 (fr)
PT (1) PT2005182E (fr)
WO (1) WO2007107714A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011038678A1 (fr) * 2009-09-29 2011-04-07 上海英伯肯医学生物技术有限公司 Méthode et stylo testeur permettant de détecter la présence de microorganismes pathogènes par immunoréaction cellulaire en réponse à une stimulation antigénique
US20110201044A1 (en) * 1998-11-04 2011-08-18 Isis Innovation Limited Tuberculosis diagnostic test
WO2012085652A3 (fr) * 2010-12-23 2012-11-22 Centre Hospitalier Universitaire Vaudois Procédés de différenciation entre des états pathologiques
WO2013037878A1 (fr) 2011-09-13 2013-03-21 Universität des Saarlandes Procédé in vitro de détermination rapide du statut infectieux d'une infection par un type de virus grippal
WO2015033136A1 (fr) 2013-09-04 2015-03-12 Imperial Innovations Limited Procédés et kits pour déterminer le statut infectieux de la tuberculose

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0618127D0 (en) * 2006-09-14 2006-10-25 Isis Innovation Biomarker
JP2009141640A (ja) * 2007-12-06 2009-06-25 Seiko Instruments Inc 電源切換回路
AU2011228996B2 (en) * 2010-03-19 2015-01-29 Lophius Biosciences Gmbh In vitro process for the quick determination of a patient's status relating to infection with Mycobacterium tuberculosis
WO2018111536A1 (fr) 2016-12-14 2018-06-21 Becton, Dickinson And Company Procédés et compositions permettant d'obtenir une évaluation de la tuberculose chez un sujet

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5736524A (en) * 1994-11-14 1998-04-07 Merck & Co.,. Inc. Polynucleotide tuberculosis vaccine
US5750356A (en) * 1996-05-31 1998-05-12 Anergen, Inc. Method for monitoring T cell reactivity
GB9624456D0 (en) * 1996-11-25 1997-01-15 Isis Innovation Assay method
CZ20031866A3 (cs) 2001-01-08 2004-03-17 Isis Innovation Limited Způsob pro stanovení účinnosti léčení mykobakteriální infekce

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BOURGARIT ANNE ET AL: "Explosion of tuberculin-specific Th1-responses induces immune restoration syndrome in tuberculosis and HIV co-infected patients." AIDS (LONDON, ENGLAND) 9 JAN 2006, vol. 20, no. 2, 9 January 2006 (2006-01-09), pages F1-F7, XP002448595 ISSN: 0269-9370 *
DELGADO JULIO C ET AL: "Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 28 MAY 2002, vol. 99, no. 11, 28 May 2002 (2002-05-28), pages 7576-7581, XP002448596 ISSN: 0027-8424 *
EMU BRINDA ET AL: "Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment." JOURNAL OF VIROLOGY NOV 2005, vol. 79, no. 22, November 2005 (2005-11), pages 14169-14178, XP002448592 ISSN: 0022-538X *
EWER K ET AL: "Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak" LANCET THE, LANCET LIMITED. LONDON, GB, vol. 361, no. 9364, 5 April 2003 (2003-04-05), pages 1168-1173, XP004784282 ISSN: 0140-6736 *
GUYOT-REVOL VALERIE ET AL: "Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis." AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 1 APR 2006, vol. 173, no. 7, 9 December 2005 (2005-12-09), pages 803-810, XP002448598 ISSN: 1073-449X *
HARARI ALEXANDRE ET AL: "Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence." JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 JAN 2005, vol. 174, no. 2, 15 January 2005 (2005-01-15), pages 1037-1045, XP002448593 ISSN: 0022-1767 *
MILLINGTON KERRY A ET AL: "Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load." JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 APR 2007, vol. 178, no. 8, 15 April 2007 (2007-04-15), pages 5217-5226, XP002448599 ISSN: 0022-1767 *
See also references of EP2005182A2 *
SEMMO NASSER ET AL: "Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection." HEPATOLOGY (BALTIMORE, MD.) MAY 2005, vol. 41, no. 5, May 2005 (2005-05), pages 1019-1028, XP002448594 ISSN: 0270-9139 *
TSUKAGUCHI K ET AL: "CD4+ alpha beta T cell and gamma delta T cell responses to Mycobacterium tuberculosis. Similarities and differences in Ag recognition, cytotoxic effector function, and cytokine production." JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 FEB 1995, vol. 154, no. 4, 15 February 1995 (1995-02-15), pages 1786-1796, XP002448597 ISSN: 0022-1767 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110201044A1 (en) * 1998-11-04 2011-08-18 Isis Innovation Limited Tuberculosis diagnostic test
US8216795B2 (en) * 1998-11-04 2012-07-10 Isis Innovation Limited Tuberculosis diagnostic test
WO2011038678A1 (fr) * 2009-09-29 2011-04-07 上海英伯肯医学生物技术有限公司 Méthode et stylo testeur permettant de détecter la présence de microorganismes pathogènes par immunoréaction cellulaire en réponse à une stimulation antigénique
WO2012085652A3 (fr) * 2010-12-23 2012-11-22 Centre Hospitalier Universitaire Vaudois Procédés de différenciation entre des états pathologiques
US20130338059A1 (en) * 2010-12-23 2013-12-19 Centre Hospitalier Universitaire Vaudois Methods for Differentiating Between Disease States
US9146236B2 (en) * 2010-12-23 2015-09-29 Giuseppe Pantaleo Methods for differentiating between disease states
WO2013037878A1 (fr) 2011-09-13 2013-03-21 Universität des Saarlandes Procédé in vitro de détermination rapide du statut infectieux d'une infection par un type de virus grippal
WO2015033136A1 (fr) 2013-09-04 2015-03-12 Imperial Innovations Limited Procédés et kits pour déterminer le statut infectieux de la tuberculose
US10041944B2 (en) 2013-09-04 2018-08-07 Mjo Innovation Limited Methods and kits for determining tuberculosis infection status
US10883990B2 (en) 2013-09-04 2021-01-05 Mjo Innovation Limited Methods and kits for determining tuberculosis infection status
US11204352B2 (en) 2013-09-04 2021-12-21 MJO Innovations Limited Methods and kits for determining tuberculosis infection status

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US20090170120A1 (en) 2009-07-02
WO2007107714A3 (fr) 2007-11-15
ES2423479T3 (es) 2013-09-20
EP2005182B1 (fr) 2013-05-01
EP2005182A2 (fr) 2008-12-24
JP5258584B2 (ja) 2013-08-07
GB0605474D0 (en) 2006-04-26
WO2007107714A8 (fr) 2009-07-16
US8765366B2 (en) 2014-07-01
AU2007228598A1 (en) 2007-09-27
PT2005182E (pt) 2013-08-02
PL2005182T3 (pl) 2013-10-31
DK2005182T3 (da) 2013-08-05
JP2009529865A (ja) 2009-08-27

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